The pathogenesis of the transmissible spongiform encephalopathies (TSE), which include Creutzfeldt Jakob disease (CJD) in humans and scrapie in sheep, remains an enigma. In this application we present evidence for the association of Spiroplasma sp., a wall-less prokaryote, with TSE. We have shown PCR amplification of Spiroplasma 16S rDNA in TSE-infected brain tissues (19) and not in control brains (0 of SO). Direct sequencing of the amplified PCR products has confirmed the presence of Spiroplasma-like DNA in all 19 of the TSE brains. Our evidence is not necessarily in conflict with involvement of a PrPres, a protease resistant host derived protein referred to as the prion, in the pathogenesis of TSE, since there is evidence that another factor is involved, which we propose to be a bacterium. Our strategy will be to optimize our probe by characterizing the entire Spiroplasma ribosomal gene in TSE and, then, screen a statistically significant number of TSE cases. In Aim 1, we will utilize a combination of universal Mollicute 16S rDNA oligonucleotide primers along with Spiroplasma-specific 16S rDNA primers to identify by PCR the near complete Spiroplasma 16S RNA gene in TSE. Direct sequencing of the PCR products will ascertain the unknown adjacent portions of the Spiroplasma-like ribosomal gene involved in TSE, from which we will design new oligonucleotide primer set/s that will detect all Spiroplasma strains, including those associated with TSE. In Aim 2 we will screen, with the new probe/s, a sizable collection of TSE cases, including 25 available CJI) human cases and 105 scrapie-infected and normal sheep brains. An additional 100 brain samples, both TSE-infected and normals, are to be sent by collaborators as randomly coded samples. The PCR amplified products in all instances will be sequenced to determine the precise nature of the Spiroplasma sp. involved. Although the role of Spiroplasma in TSE cannot be determined from these experiments, the presence of this microbe in all cases of TSE and not in controls would provide the basis for developing a test for TSE